Genome-Wide Identification and Characterization of the Trehalose-6-phosphate Synthetase (TPS) Gene Family in Watermelon (Citrullus lanatus) and Their Transcriptional Responses to Salt Stress

With the increase in watermelon cultivation area, there is an urgent need to explore enzymatic and genetic resources for the sustainable development of watermelon, especially under salt stress. Among the various compounds known, trehalose plays an important role in regulating abiotic stress tolerances in diverse organisms, including plants. Therefore, the present study comprehensively analyzed the trehalose-6-phosphate synthase (TPS) gene family in watermelon. The study analyzed the functional classification, evolutionary characteristics, and expression patterns of the watermelon TPS genes family. Seven ClTPSs were identified and classified into two distinct classes according to gene structure and phylogeny. Evolutionary analysis suggested the role of purifying selection in the evolution of the TPS family members. Further, cis-acting elements related to plant hormones and abiotic stress were identified in the promoter region of the TPS genes. The tissue-specific expression analysis showed that ClTPS genes were widely expressed in roots, stems, leaves, flowers, and fruits, while ClTPS3 was significantly induced under salt stress. The overexpression of ClTPS3 in Arabidopsis thaliana significantly improved salt tolerance. Finally, the STRING functional protein association networks suggested that the transcription factor ClMYB and ClbHLH regulate ClTPS3. Thus, the study indicates the critical role of ClTPS3 in watermelon response to salt stress.


Introduction
Trehalose (α-D-glucopyranosyl-1, 1-α-D-glucopyranoside) is a non-reducing disaccharide consisting of two glucose molecules linked by α, α, 1, 1-glycosidic bonds [1]. It has a symmetrical structure, with two glucose molecules having symmetry more stable than maltose, sucrose, glucose, and other sugars of small molecules [2,3]. Therefore, the physical and chemical properties of trehalose are different from its analogs, making it an important component in keeping cells alive. Trehalose is known as a living substance, which exists in all living organisms and plays a major role in plant growth and development [4,5]. In recent years, trehalose has attracted extensive attention as a potential signal metabolite and a cell stabilizer of plants. Cells exposed to high temperature, freezing temperature, radiation, drought, high osmotic pressure, high salinity, and other adverse environmental conditions synthesize trehalose in large quantities, which plays an important role in maintaining osmotic pressure, protecting membrane structure, and participating in the signal transduction process. Subsequently, once the crisis is resolved, trehalose decomposes rapidly to substances that act as energy sources [2].
Trehalose in plants was first identified in Selaginella lepidophylla (Hook. and Grev.) Spring almost 100 years ago. A previous study found that high trehalose levels in Selaginella helped it to survive under an extreme drought environment [3]. However, increasing farmers' income in China. However, soil salinization is a major problem affecting the production and quality of watermelon. According to survey statistics, about one billion hm 2 of land worldwide is affected by salinization [36]. It is estimated that more than 50% of the global arable land area will be salinized by 2050 [37]. The area of salinized soil in China is thirty-six million hm 2 , accounting for 4.88% of the available land area, mainly distributed in the north and the coastal regions [38,39]. Studies have shown that most plants are damaged in soils with a salt content of up to 0.3% [40]. Salt stress mainly inhibits plant growth. However, with the aggravation of salt stress, the leaf area stops increasing, and the aboveground and underground fresh and dry weight decreases significantly [41]. Salt stress can either directly inhibit plant growth or indirectly affect plant growth by inhibiting photosynthesis and reducing the synthesis of growth substances. Moreover, the higher the salt concentration, the longer the action time, and the more noticeable the inhibition effect [42]. For watermelon, salt stress decreases planting area, yield, and quality [43]. Furthermore, with the annual expansion of the watermelon cultivation area, soil salinization will become more serious, which will have a serious impact on the sustainable and healthy development of watermelon.
Therefore, the present study investigated the functional classification, evolutionary characteristics, and expression profile of the ClTPS family. The study on the ClTPS gene will help breeders effectively select high-quality salt-tolerant germplasm resources to maintain watermelon production under adverse conditions. The study will also be of great significance for the transformation and utilization of saline-alkali land and the improvement of agricultural production levels.

Identification of Watermelon TPS Genes and Distribution of TPS Proteins in Plant
Research has shown that TPS proteins contain two conserved domains, TPS and TPP [13,44]. Seven ClTPSs were identified from the watermelon genome database Cu-GenDB (http://cucurbitgenomics.org/organism/21) (accessed on 20 Octomber 2021) by BLASTP and numbered from ClTPS1 to ClTPS7 depending on their location on the chromosome ( Table 1). The length of most of these TPS proteins (71.4%) ranged from 831 to 860 amino acids (aa), while the largest TPS (ClTPS2) had 933 aa and the smallest (ClTPS1) had 831 aa. The molecular weight (MW) ranged from 94.10 to 105.37 kDa and the predicted isoelectric points (pI) from 5.57 to 6.46. Finally, subcellular localization prediction indicated that the seven ClTPS proteins were located in the vacuole, including four showing chloroplast localization (ClTPS1, ClTPS4, ClTPS 6, and ClTPS7) and two showing cytoplasm localization (ClTPS2 and ClTPS3) too. Meanwhile, the seven ClTPSs were located in seven different chromosomes (chromosomes 1, 3, 5, 6, 7, 10, and 11) of watermelon ( Figure 1A), and a gene duplication event was detected.
Furthermore, a collinear relationship diagram of the dicots watermelon, A. thaliana, and melon TPS family was constructed to further understand the evolutionary mechanism of the watermelon TPS family ( Figure 1B). Eight pairs of direct homologous genes were identified between watermelon and A. thaliana, and nine between watermelon and melon. Collinearity analysis detected ClTPS3, ClTPS4, ClTPS6, and ClTPS7 in the three plants, suggesting that these genes may be highly conserved. In addition, the K a /K s ratio of the eight pairs of direct homologous genes between watermelon and A. thaliana were less than 0.1 except for ClTPS7/AtTPS2 (supplementary Table S1), indicating purifying selection as the main driving force for the evolution of the watermelon TPS genes.
Furthermore, a collinear relationship diagram of the dicots watermelon, A. thaliana, and melon TPS family was constructed to further understand the evolutionary mechanism of the watermelon TPS family ( Figure 1B). Eight pairs of direct homologous genes were identified between watermelon and A. thaliana, and nine between watermelon and melon. Collinearity analysis detected ClTPS3, ClTPS4, ClTPS6, and ClTPS7 in the three plants, suggesting that these genes may be highly conserved. In addition, the Ka/Ks ratio of the eight pairs of direct homologous genes between watermelon and A. thaliana were less than 0.1 except for ClTPS7/AtTPS2 (supplementary Table S1), indicating purifying selection as the main driving force for the evolution of the watermelon TPS genes.

Phylogenetic Analysis, Structural and Conserved Motifs of ClTPSs
Seventy TPS proteins sequences from seven species and seven ClTPSs were used to construct a phylogenetic tree to understand the evolutionary relationship and classification of ClTPSs ( Figure 2). All TPS proteins of watermelon were classified into two groups: Class I and Class II. Class I harbored two members, including ClTPS2 and ClTPS7; Class II harbored five members, including ClTPS1, ClTPS3, ClTPS4, ClTPS5, and ClTPS6. Phylogenetic analysis indicated that the protein was divided into two categories based on their sequences ( Figure 3A). Generally, the most closely related members of each group

Phylogenetic Analysis, Structural and Conserved Motifs of ClTPSs
Seventy TPS proteins sequences from seven species and seven ClTPSs were used to construct a phylogenetic tree to understand the evolutionary relationship and classification of ClTPSs ( Figure 2). All TPS proteins of watermelon were classified into two groups: Class I and Class II. Class I harbored two members, including ClTPS2 and ClTPS7; Class II harbored five members, including ClTPS1, ClTPS3, ClTPS4, ClTPS5, and ClTPS6. Phylogenetic analysis indicated that the protein was divided into two categories based on their sequences ( Figure 3A). Generally, the most closely related members of each group had a similar exonintron structure, with little difference in the length of introns and exons. Analysis of the exon-intron organization showed that all members except ClTPS1 in group II contain 3 exons, while group I members have 17 exons ( Figure 3B). had a similar exon-intron structure, with little difference in the length of introns and exons. Analysis of the exon-intron organization showed that all members except ClTPS1 in group II contain 3 exons, while group I members have 17 exons ( Figure 3B). Furthermore, MEME software was used to search for motifs and determine the predicted structural characteristics of ClTPS proteins ( Figure 3C). Interestingly, all the members in group II contained 18 motifs, except ClTPS4 lacking motif 20; members of group I had only 14 motifs (lacking 5/8/9/14/15/20 motifs).

Figure 2.
Phylogenetic relationship of TPS proteins among watermelon and other seven species. All TPS proteins were divided into two subgroups, represented by two colors. The green color represents Class I protein, and the blue color represents Class II protein. The phylogenetic tree was constructed by MEGA7 software with 1000 bootstrap replicates, following the neighbor-joining method. had a similar exon-intron structure, with little difference in the length of introns and exons. Analysis of the exon-intron organization showed that all members except ClTPS1 in group II contain 3 exons, while group I members have 17 exons ( Figure 3B). Furthermore, MEME software was used to search for motifs and determine the predicted structural characteristics of ClTPS proteins ( Figure 3C). Interestingly, all the members in group II contained 18 motifs, except ClTPS4 lacking motif 20; members of group I had only 14 motifs (lacking 5/8/9/14/15/20 motifs).

Figure 2.
Phylogenetic relationship of TPS proteins among watermelon and other seven species. All TPS proteins were divided into two subgroups, represented by two colors. The green color represents Class I protein, and the blue color represents Class II protein. The phylogenetic tree was constructed by MEGA7 software with 1000 bootstrap replicates, following the neighbor-joining method. Furthermore, MEME software was used to search for motifs and determine the predicted structural characteristics of ClTPS proteins ( Figure 3C). Interestingly, all the members in group II contained 18 motifs, except ClTPS4 lacking motif 20; members of group I had only 14 motifs (lacking 5/8/9/14/15/20 motifs).

Prediction of Cis-Acting Elements of the ClTPS Genes
The 2000 bp sequences upstream of the start site of seven ClTPSs were used to identify the potential cis-acting elements in the promoter region for abiotic/biotic stress. The method predicted 249 cis-acting elements in these genes ( Figure 4, Table S2). Many cis-acting elements were involved in response to environmental stress, hormone-responsiveness, development, light response, site binding, and other functions ( Figure 4A). The most abundant elements were environmental stress-related elements, including those involved in lowtemperature and anaerobic induction; all genes except CTPS7 contained low-temperature responsiveness elements, indicating that they may be considerably affected by the ambient temperature. Meanwhile, ABRE (ABA) was the most abundant among the predicted hormone-responsive elements ( Figure 4B, Table S2).

Prediction of Cis-Acting Elements of the ClTPS Genes
The 2000 bp sequences upstream of the start site of seven ClTPSs were used to identify the potential cis-acting elements in the promoter region for abiotic/biotic stress. The method predicted 249 cis-acting elements in these genes ( Figure 4, Table S2). Many cisacting elements were involved in response to environmental stress, hormone-responsiveness, development, light response, site binding, and other functions ( Figure 4A). The most abundant elements were environmental stress-related elements, including those involved in low-temperature and anaerobic induction; all genes except CTPS7 contained low-temperature responsiveness elements, indicating that they may be considerably affected by the ambient temperature. Meanwhile, ABRE (ABA) was the most abundant among the predicted hormone-responsive elements ( Figure 4B, Table S2).

Tissue-Specific Expression of ClTPS Genes
Initially, the transcript profiles derived from the NCBI database were used to analyze the expression levels of ClTPSs in watermelon flowers, fruits, stems, leaves, and roots to

Tissue-Specific Expression of ClTPS Genes
Initially, the transcript profiles derived from the NCBI database were used to analyze the expression levels of ClTPSs in watermelon flowers, fruits, stems, leaves, and roots to elucidate their functions and provide the basis for further understanding of the tissuespecific expression pattern ( Figure 5). The seven ClTPSs were divided into two groups; the genes clustered in the same group had a similar expression pattern; for example, ClTPS1/2/7 had low expression levels in fruits and leaves, with no expression of ClTPS1 in fruits. The other ClTPSs were widely expressed in all tissues, with ClTPS5 and ClTPS6 showing relatively high expression levels throughout development. elucidate their functions and provide the basis for further understanding of the tissuespecific expression pattern ( Figure 5). The seven ClTPSs were divided into two groups; the genes clustered in the same group had a similar expression pattern; for example, ClTPS1/2/7 had low expression levels in fruits and leaves, with no expression of ClTPS1 in fruits. The other ClTPSs were widely expressed in all tissues, with ClTPS5 and ClTPS6 showing relatively high expression levels throughout development.

Expression of ClTPS Genes under Different Salt Stress
To further explore the role of ClTPSs in salt stress response, the expression levels of ClTPSs under different NaCl concentrations, including 0, 50, 100, 150, 200, and 250 mM, and at various time points, including 0, 0.5, 6, 24, 48, and 72 h, under 200 mM NaCl were analyzed by qRT-PCR results (Figures 6 and 7). Under different NaCl concentrations, these genes (ClTPS2 had no value) showed similar expression patterns ( Figure 6). In the concentration range 0-150 mM, the expression levels of the six genes gradually increased and then decreased at 200-250 mM concentration range.
Further analysis of genes at different time points under 200 mM NaCl revealed that six genes showed a decrease towards 0.5 h, an increase from 0.5 to 24 h, and then a decrease from 24 to 72 h; the expression level peaked at 24 h ( Figure 7). Dehydration, wilting, and death of watermelon seedlings under high osmotic conditions probably resulted in this expression pattern. Meanwhile, ClTPS3 showed the highest expression level among all treatments, suggesting it is a key gene involved in salt stress response.

Expression of ClTPS Genes under Different Salt Stress
To further explore the role of ClTPSs in salt stress response, the expression levels of ClTPSs under different NaCl concentrations, including 0, 50, 100, 150, 200, and 250 mM, and at various time points, including 0, 0.5, 6, 24, 48, and 72 h, under 200 mM NaCl were analyzed by qRT-PCR results (Figures 6 and 7). Under different NaCl concentrations, these genes (ClTPS2 had no value) showed similar expression patterns ( Figure 6). In the concentration range 0-150 mM, the expression levels of the six genes gradually increased and then decreased at 200-250 mM concentration range.
Further analysis of genes at different time points under 200 mM NaCl revealed that six genes showed a decrease towards 0.5 h, an increase from 0.5 to 24 h, and then a decrease from 24 to 72 h; the expression level peaked at 24 h ( Figure 7). Dehydration, wilting, and death of watermelon seedlings under high osmotic conditions probably resulted in this expression pattern. Meanwhile, ClTPS3 showed the highest expression level among all treatments, suggesting it is a key gene involved in salt stress response.

Functional Analysis of ClTPS3 Gene
The above results proved that salt stress significantly induced ClTPS3. A genetic transformation experiment was carried out in A. thaliana, and the plants were grown under 200 mM NaCl stress to verify the function of ClTPS3 further. The results showed that the expression level of ClTPS3 in transgenic plants OE1, OE2, and OE3 was significantly higher than that of the wild-type plants (CK), which was 7.4, 7.8 and 8.2 times higher than CK, respectively ( Figure 1).

Functional Analysis of ClTPS3 Gene
The above results proved that salt stress significantly induced ClTPS3. A genetic transformation experiment was carried out in A. thaliana, and the plants were grown under 200 mM NaCl stress to verify the function of ClTPS3 further. The results showed that the expression level of ClTPS3 in transgenic plants OE1, OE2, and OE3 was significantly higher than that of the wild-type plants (CK), which was 7.4, 7.8 and 8.2 times higher than CK, respectively ( Figure 1).

Functional Analysis of ClTPS3 Gene
The above results proved that salt stress significantly induced ClTPS3. A genetic transformation experiment was carried out in A. thaliana, and the plants were grown under 200 mM NaCl stress to verify the function of ClTPS3 further. The results showed that the expression level of ClTPS3 in transgenic plants OE1, OE2, and OE3 was significantly higher than that of the wild-type plants (CK), which was 7.4, 7.8 and 8.2 times higher than CK, respectively ( Figure 1). The transgenic plants grew normally under high salt concentration, while the growth of CK was significantly inhibited ( Figure 8B). The transgenic plants had a root length of 35.19, 36.92, and 39.03 mm, respectively, which was three times more than that of CK ( Figure 8C). To investigate the salt tolerance of transgenic plants and CK, the seedlings were treated with 200 mM NaCl for one week, and the results showed that transgenic plants had less wilting and a higher survival rate than CK ( Figure 8C). To confirm this result, trehalose content, fresh weight (FW), dry weight (DW), and relative water content (RWC) were measured. The results showed that the trehalose content of transgenic plants was significantly higher than that of CK ( Figure 8E), and the data of FW, DW and RWC exhibited similar trends ( Figure 8F-H). In addition, the MDA content of the CK was significantly higher than that of transgenic plants, while POD activity and SOD activity were significantly lower than that of transgenic plants ( Figure 8I-K), which indicates that ClTPS3-overexpressed reduced the content of H 2 O 2 and O 2 − and reduced the cell membrane damage. These results suggest that the ClTPS3 gene may improve salt tolerance. 35.19, 36.92, and 39.03 mm, respectively, which was three times more than that of CK (Fig  ure 8C). To investigate the salt tolerance of transgenic plants and CK, the seedlings were treated with 200 mM NaCl for one week, and the results showed that transgenic plant had less wilting and a higher survival rate than CK ( Figure 8C). To confirm this result trehalose content, fresh weight (FW), dry weight (DW), and relative water content (RWC were measured. The results showed that the trehalose content of transgenic plants wa significantly higher than that of CK ( Figure 8E), and the data of FW, DW and RWC exhib ited similar trends ( Figure 8F-H). In addition, the MDA content of the CK was signifi cantly higher than that of transgenic plants, while POD activity and SOD activity were significantly lower than that of transgenic plants ( Figure 8I-K), which indicates tha ClTPS3-overexpressed reduced the content of H2O2 and O2− and reduced the cell mem brane damage. These results suggest that the ClTPS3 gene may improve salt tolerance.

Functional Protein Association Networks of ClTPS Genes
As mentioned above (Figure 4), many MYB and bHLH transcription factor binding sites were detected in the promoter region of ClTPS genes, indicating their regulation by the transcription factors. A functional protein association network analysis was performed to screen for transcription factors that regulate ClTPS genes. Studies have reported the roles of MYB and bHLH transcription factor family genes in regulating watermelon response to abiotic stress [45,46]. Therefore, the interactions among the TPS proteins, MYB, and bHLH transcription factors were predicted using the STRING program. The analysis indicated a direct regulatory relationship between TPS9 (ClTPS3) and bHLH093 (ClbHLH93), while MYB36 (ClMYB9), MYB78 (ClMYB69), and MYB79 (ClMYB51) appeared to regulate bHLH093 ( Figure 9). ical replicates. Different letters indicate significant differences within treatments by AN 0.05).

Discussion
The present study adopted a bioinformatic approach to identify and charact watermelon TPS gene family. Seven ClTPS genes were identified in watermelo was less than the number of genes in A. thaliana and rice (11 TPS gene members). studies revealed that fragment duplication resulted in three TPS genes in rice, wide duplication produced one TPS gene in A. thaliana; AtTPS2 and AtTPS3 ge produced by fragment duplication from AtTPS1 [44]. However, in this study, no duplication or fragment duplication event was detected in the ClTPS gene family ing different ancestor genes for these seven ClTPSs. This observation confirmed AtTPS7 (At1g06410.1, ClTPS5, Cla97C07G130930.1); AtTPS8 (At1g70290.1, ClTPS3, Cla97C05G107320.1); AtTPS9 (At1g23870.1, ClTPS3, Cla97C05G107320.1). Blue lines represent gene interaction confidence (0 to 1); thick lines indicate confidence score higher than 0.85, while thin lines indicate confidence score between 0.26 and 0.84.

Discussion
The present study adopted a bioinformatic approach to identify and characterize the watermelon TPS gene family. Seven ClTPS genes were identified in watermelon, which was less than the number of genes in A. thaliana and rice (11 TPS gene members). Previous studies revealed that fragment duplication resulted in three TPS genes in rice, genomewide duplication produced one TPS gene in A. thaliana; AtTPS2 and AtTPS3 genes were produced by fragment duplication from AtTPS1 [44]. However, in this study, no tandem duplication or fragment duplication event was detected in the ClTPS gene family, indicating different ancestor genes for these seven ClTPSs. This observation confirmed the presence of at least seven TPS genes in the common ancestor of monocotyledonous and dicotyledonous plants [47]. Meanwhile, in genetics, the K a /K s ratio is used as an indicator of selective pressure (the force applied by natural selection) acting on a protein-coding gene. K a /K s greater than 1 indicates a strong positive selection, genes with K a /K s between 0.5 and 1 have a weak positive selection, and those with K a /K s less than 1 are negatively selected (purifying selection) [48][49][50]. In this study, the analysis of evolutionary selective pressure indicated that the ClTPS genes were subjected to a purifying selection, which may partly explain fewer genes than A. thaliana.
According to the gene structure and enzyme activity, TPS family genes in plants are classified into two classes: Class I and Class II [51][52][53][54][55]. In the phylogenetic tree, ClTPS2 and ClTPS7 clustered with AtTPS1, AtTPS2, AtTPS3, AtTPS4, and OsTPS1, belonging to the Class I TPS subfamily, while the other five ClTPS genes belonged to the Class II TPS subfamily. Studies have also shown that the Class I genes have TPS activity, while the function of Class II TPS subfamily genes is not clear [52,[56][57][58]. However, few researchers pointed out that the N-terminal of some genes in Class II TPS subfamily have the TPS domain of the conserved sites of glycotransferase and the TPP domain of the conserved sites of two phosphohydrolases at the C-terminal; the presence of these two domains indicate that the gene members are either bifunctional or act as a TPS complex subunit [54,55,59]. Long et al. [28] predicted ten glycosylation sites and forty-nine phosphorylation sites for the protein encoded by the ClTPS1 gene, indicating TPS and TPP activities for the ClTPS1 gene. The functional verification of the TPS gene family in maize revealed both TPS activity and TPP activity for the ZmTPS3 gene of the Class II subfamily [60]. These observations collectively indicate that the TPS genes may quickly respond to stress and synthesize trehalose to help watermelon survive under stress.
The ClTPS genes of Class I and Class II subfamily had significantly different numbers of exons, indicating evolutionary differences between them similar to Populus, A. thaliana, and rice [44]. Studies have proven a close relationship between the structure and function of genes [61]. Similarly, the differences in the expression patterns and functions of Class I and Class II ClTPS genes may be related to the wide differences in the exon-intron structure. In this study, two Class I members and four Class II members showed similar tissue expression patterns ( Figure 5B), probably because few introns are required during the selective splicing of mRNA to subsequently regulate the structure and function of the protein encoded by the gene. Meanwhile, some genes may have enhanced mRNA transcription and transport, leading to different tissue-specific patterns and different expression levels [62,63].
TPS is a gene that encodes a key enzyme in the trehalose pathway. The promoter region of ClTPS genes contains a variety of signal response elements, which can respond to various stress conditions. Moreover, each ClTPS gene contains different stress response elements, indicating that different signals may induce each gene. The ClTPS5 gene has the largest number of hormone-responsive cis-acting elements, consistent with the highest expression level in the tissues. Many cis-acting elements involved in drought-inducibility and anaerobic induction were identified in the ClTPS3 gene, in agreement with the high expression level under osmotic stress. Studies have demonstrated that an important feature of drought and salt stress is cell osmosis, which leads to ABA accumulation as an adaptive response [64]. Meanwhile, the relationship between ScTPS and ABA signals in sugarcane indicated the role of ScTPS in drought tolerance [65]. Most of the ClTPS genes also had ABA cis-acting elements, suggesting their roles in the drought stress response.
Studies have shown that high temperature, high salt concentration, and drought stress significantly increased the expression levels of TPS1 and TPS7 genes in potatoes, suggesting the role of the TPS gene in the signal transduction pathway of stress tolerance [13]. Similarly, the expression level of PhTPS6 in petunia was significantly upregulated after 12 h of low-temperature treatment, with a gradual increase in the expression level with the treatment time [66]. PhTPS6 showed the same expression pattern after NaCl treatment, suggesting its role under low temperature and salt stress [66]. A recent study in sugarcane showed that salt and drought induced the expression of the ScTPS1 gene, which indicated that sugarcane maintained the cell osmotic stability by increasing trehalose-6-phosphate production and alleviated the simulated stress. These observations suggested that genetic engineering of endogenous ScTPS1 gene involved in trehalose biosynthesis may improve the drought tolerance of sugarcane [13]. Similarly, salt stress markedly induced ClTPS3 (Figures 6 and 7). Previous studies had proved that trehalose could improve the salt tolerance of plants by increasing the activity of antioxidant enzymes, and it was found that the activities of SOD, CAT, POD were positively correlated with the salt tolerance of plants [29,67]. In addition, trehalose significantly reduced the accumulation of MDA in tomato, indicating that trehalose can reduce the degree of cell membrane damage and improve the salt tolerance of tomato [68]. In this study, the trehalose content of ClTPS3 transgenic A.thaliana plants was significantly increased, and the activities of SOD and POD were also significantly increased. In addition, the MDA content was significantly decreased, while the relative water content was significantly increased, indicating that ClTPS3 played an important role in the salt tolerance of watermelon. Furthermore, the results of STRING functional protein association networks showed that ClMYB and ClbHLH transcription factors most likely regulated only ClTPS3 among the seven ClTPS genes. These results indicate a putative role of ClTPS3 in watermelon response to various abiotic stresses. Therefore, effective use of the ClTPS3 gene may help watermelon maintain growth and production under salt stress.

Chromosomal Localization, Phylogenetic Analysis, Duplication Events, and Collinearity Analysis of ClTPS Genes
The chromosomal distribution of ClTPSs was derived from CuGenDB. The TBtools software [72] was used to determine the chromosome localization and the ClTPSs duplication events. Furthermore, 77 protein sequences of various species, including watermelon, Populous, Cucumis melo, Malus domestica, A. thaliana, Oryza sativa, Glycine max, and Triticum aestivum, were used to construct the phylogenetic tree. Amino acid sequence alignment was carried out using Clustal W [73], and then the phylogenetic trees were constructed following the neighbor-joining (NJ) method with 1000 bootstrap using the MEGA 7.0 software [74]. TPS genes duplication events, collinearity, and selective evolutionary pressure were analyzed using the TBtools software [72].

Analysis of ClTPS Gene Structures and Conserved Motifs
The structure (exon-intron arrangement) of ClTPS genes was obtained from CuGenDB and visualized using TBtools. Then, the putative conserved motifs of ClTPS protein sequences were analyzed using the MEME program (http://meme-suite.org/tools/meme) (accessed on 8 June 2021), with any number of repetitions, a maximum of 20 motifs; and an optimum width of each motif between 6 and 60 residues [75]; the conserved domains were visualized using TBtools.

Analysis of Cis-Acting Elements
TBtools was used to extract the 2000 bp sequences upstream of the transcriptional start site (ATG) of the ClTPS genes as the putative promoter region; these sequences were submitted to Plantcare (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) (accessed on 8 June 2021) to identify the cis-acting elements in the promoter region.

Tissue-Specific Expression Patterns and Expression Levels under Different Stresses of ClTPS Genes
The data of different tissues, including flower (GSE69073), fruit (PRJNA338036), stem (SRP012853), leaf (PRJNA381300), and root (PRJNA641525), and under different stresses, such as drought (GSE144814), osmotic stress (PRJNA381300), heat shock (PRJNA504354) and cold (PRJNA328189), were retrieved from the NCBI database (https://www.ncbi.nlm.nih.gov/) (accessed on 22 June 2021). The data were first downloaded from the SRA database, converted into fastq format, and uploaded into the Kallisto Super Wrapper of TBtools to obtain the transcript expression matrix. Finally, the gene expression matrix was obtained from the transcript expression matrix using the Trans Value Sum of TBtools. Furthermore, hierarchical clustering and heatmap of ClTPS genes were generated using TBtools.

Plant Growth and Treatments
Watermelon ("HQ-2" variety) seedlings were maintained in Hoagland solution in a growth chamber at 25 • C under a photoperiod of 16 h light/8 h dark. After growing for 30 days, the seedlings were grown hydroponically in Hoagland solution containing 0 (control), 50, 100, 150, 200, or 250 mM NaCl; thirty seedlings were maintained per treatment. Control plants (were cultured in standard Hoagland solution) were cultured in parallel. Seedlings were sampled after 3 days of salt treatment. In addition, samples under 200 mM NaCl were collected at 0, 0.5, 6, 24, 48, and 72 h. Finally, all the samples were immediately frozen in liquid nitrogen and stored at −80 • C until for RNA extraction. Three seedlings were mixed into one sample, and three biological replicates were maintained per treatment.

RNA Extraction and Quantitative Real-Time-PCR
Total RNA was isolated using the Plant RNA Kit (Huayueyang, Beijing, China) according to the manufacturer's protocol. The first strand of cDNA was obtained using a PrimeScript RT reagent Kit (TaKaRa, Dalian, China). qRT-PCR was performed on the Light Cycler480 Real-Time System (Bio-Rad Laboratories) with the method described previously [61]. ClActin was used as the reference gene, and the specific primers are shown in Supplementary Table S3. The data were analyzed using the 2 −∆∆Ct method [76].

Functional Analysis of ClTPS3 Gene
The coding sequence of ClTPS3 (Cla97C05G107320.1) was inserted into the plant transformation vector pRI101-AN at Nde I-Kpn I sites to generate the ClTPS3-overexpressing recombinant vector, and the specific primer sequences used for PCR amplification were as follows: 5 -CATATGATGGCATCAAGATCCCCCAC-3 and 5 -GGTACCTCAAAAAACACTC TCAAAAGAAACAC-3 . The construct was transformed into Agrobacterium tumefaciens strain GV3101.
The Agrobacterium dipping flower method was employed to infect A. thaliana [77]. The seeds of the overexpressed homozygote and the wild-type A. thaliana were disinfected and seeded on the MS medium and cultured under normal light. The overexpressed (OE) and wild-type A.thaliana (CK) plants were transferred to an MS medium containing 200 mM NaCl for stress treatment. After 7 days, the root length was measured using a vernier caliper (SHRN 0-300 mm, Guilin, China), and at least ten seedlings were measured for each line. In addition, the 3-week-old OE and CK seedlings were treated with 200 mM NaCl for 7 days, then three seedlings were sampled for the fresh weight (FW), dry weight (DW) and relative water content (RWC) measurement, and ten seedlings were immediately frozen in liquid nitrogen and stored at −80 • C until for RNA extraction and indexes analysis. Furthermore, the trehalose, MDA, POD, and SOD content were determined using BC0330 (Solarbio, Beijing, China), KTB1050, KTB1150, and KTB1030 (Abbkine, Beijing, China) kits, respectively. The absorbance was measured using the SpectraMax i3X Multimode Detection Platform Molecular Devices (Molecular Devices, China).
All data were statistically analyzed using three biological replicates and expressed as mean values ± standard deviation (SD). SPSS 18.0 statistical software was used for variance analysis, and differences were considered statistically significant at a p-value of 0.05 (p < 0.05).

Functional Protein Association Networks of ClTPS Genes
A TPS-related functional protein association network was built using the online software STRING (http://stringdb.org) (accessed on 1 May 2021) using the TAIR gene ID of genes related to TPS. The amino acid sequences of the MYB transcription factors and bHLH transcription factors were derived from the articles of Wang [45] and He [46]. The gene ID of A. thaliana may be responsible for multiple members in watermelon. The transcription factors with the highest amino acid identity were selected (Table S4). The confidence level of minimum required interaction score parameters was set at 0.25.

Conclusions
In this study, a total of seven TPS genes were identified through genome-wide analysis in watermelon, divided into two groups, and located on seven chromosomes. Tissuespecific expression and stress response analysis showed diversity and specificity in the expression patterns of ClTPSs in different tissues and under various stresses. The ClTPS3 gene was highly expressed under salt stress, suggesting an essential role in watermelon's response to salt stress. Our results will help to lay a foundation for further understanding the structures and characteristics of the TPS gene family and improving the efficiency of watermelon breeding.